The route of cellular entry for most conventional drugs is diffusion across the biological membrane. For this reason, drugs tend to be small (MW<500) and amphipathic, containing both hydrophobic and hydrophilic functionalities. These characteristics engender molecules with water solubility, while allowing them to cross the nonpolar lipid bilayer of the cell membrane. In contrast, the drugs used in antisense and gene therapies are relatively large hydrophilic polymers and are frequently highly negatively charged as well. Both of these physical characteristics preclude their direct diffusion across the cell membrane. For this reason, the major barrier to gene therapy and antisense therapy is the delivery of the drug to the cellular interior. This situation is in contrast to standard drug development in which the identification of the drug is the major barrier in development.
Gene or polynucleotide transfer to cells is an important technique for biological and medical research as well as potentially therapeutic applications. The polynucleotide needs to be transferred across the cell membrane and into the cell. Gene transfer methods currently being explored include viral vectors and non-viral methods.
Viral delivery was first accomplished with mouse retroviruses. However, these vectors cannot infect all cell types efficiently, especially in vivo. Therefore, several viral vectors, including Herpes virus, Adenovirus, Adeno-associated virus and others are being developed to enable more efficient gene transfer different cell types.
For non-viral delivery, polynucleotides can be incorporated into lipid vesicles (liposomes) or complexed with polymers. Other non-viral methods of polynucleotide delivery to cells include electroporation and “gene gun” technologies. One of the several methods of polynucleotide delivery to cells is the use of polynucleotide/polycation complexes. It has been shown that cationic proteins, like histones and protamines, or synthetic polymers, like polylysine, polyarginine, polyomithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular polynucleotide delivery agents.
Polycations facilitate nucleic acid condensation. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+, Fe3+, and natural or synthetic polymers such as histone H1, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized.
The volume which one polynucleotide molecule occupies in a complex with polycations is much lower than the effective volume of the free polynucleotide molecule. The size of a polynucleotides/polymer complex is probably critical for gene delivery in vivo and possible for in vitro as well. For intravascular delivery, the polynucleotide needs to cross the endothelial barrier in order to reach the parenchymal cells of interest. The largest endothelial fenestrae (holes in the endothelial barrier) occur in the liver and have an average diameter of 100 nm. The trans-epithelial pores in other organs are much smaller. For example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm. The size of the polynucleotide complexes is also important for the cellular uptake process. After binding to the cells the polynucleotide/polycation complex is likely taken up by endocytosis. Since endocytic vesicles have a typical internal diameter of about 100 nm, polynucleotide complexes smaller than 100 nm are preferred. The compacted form of the condensed polynucleotide/polycation complexes also protects the polynucleotide from nuclease degradation, both in serum and in acidic intracellular environments.
Polycations may provide attachment of polynucleotides to the cell surface. The polymer forms a cross-bridge between the polyanionic polynucleotide and the polyanionic surface of a cell. As a result, the mechanism of polynucleotide translocation to the intracellular space might be non-specific adsorptive endocytosis. Polycations also provide a convenient linker for attaching specific ligands to the complex, thereby allowing targeting to specific cell types.
Polymers can also facilitate cellular entry of polynucleotides. For instance, some polymers, such as polyethylenimine, are thought to probably disrupt endosomal/lysosomal function through a proton sponge effect. Disruption of endosomal/lysosomal function has also been accomplished by linking endosomal or membrane disruptive agents such as fusion peptides or adenoviruses to the polycation or complex. Polymers that are pH-sensitive have found broad application in the area of drug delivery because of their ability to exploit various physiological and intracellular pH gradients for the purpose of controlled release of drugs. pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over a range of pH. Narrower definitions demand significant changes in the polymer's ability to retain or release a bioactive substance in a physiologically tolerated pH range (typically pH 5.5-8).